The attachment of N-acetylglucosamine residue through an -O- linkage to serine/threonine residues of proteins is a recently discovered novel form of protein glycosylation. This type of glycosylation is found only on cytosolic and nuclear proteins of mammalian cells. In contrast, the classical forms of O- or N-linked glycosylation consists of large oligosaccharide structures found on externally oriented plasma membrane proteins. O-linked N-acetylglucoseamine glycosylation is dynamic in that cells have enzymes to attach and remove the sugar residue in response to yet unknown second messengers. This system may be analogous to the second messenger signaling via kinases and phosphatases. We searched for O-linked GlucNAc glycosylation in platelets by specifically labelling the GlucNAc residues with the enzyme galactosyl transferase and with radioactively labelled UDP-galactose. In total lysates of resting platelets four proteins with MW of 152, 122, 79 and 52 kDa were labelled. Subcellular fractionation localized the 122 and 79 kDa proteins to the cytosol. Using wheat germ affinity chromatography we were able to isolate O-GlucNac proteins from platelet cytosol. These proteins will now be analyzed by obtaining partial amino acid sequences to match against known protein sequences. Identity of these proteins will be helpful in determining the function of O-linked GlucNAc in platelets. The significance of this project lies in identification of new biochemical pathways of platelet activation which could potentially be used to modify platelet reactiveness in vivo or in vitro.